There is a need for more rapid ways of measuring and monitoring the presence and concentration of analytes in biological samples for example in the diagnosis or monitoring of medical conditions or physiological states in biological systems.
In the field of medicine, the point of sampling from a human or animal subject is often also a point of care, for example the subject is in the presence of a health professional ready to take steps depending on the result of the analytical outcome. The time taken for a biological sample to be transported from its collection point, say a point of care (e.g. doctor surgery or clinic, hospital bedside, operating theatre), to a suitable laboratory or dedicated laboratory area can amount to significant delay. With each additional handling and transport step taking several minutes or longer, it can be a matter of hours or more before a relevant result or reading is available from any given sample, especially where analytical techniques requiring further sample preparation e.g. HPLC are involved.
Biosensors are valuable tools in the measurement of a wide range of analytes in biological samples. A typical biosensor comprises a sensing electrode having a biorecognition molecule, such as an antibody or enzyme, attached to a transducer so as to enable detection of the binding of an analyte to the biorecognition molecule. Examples of transducers for biosensors include electrochemical, piezoelectric, optoelectronic, fibre optic, thermistor, diode or acoustic devices. A wide range of compounds and polymers can be used to enable the transfer of electrons between a suitable biorecognition molecule and an electrically conductive substrate.
Entrapment of biorecognition molecules in electropolymer matrices has enabled the construction of biosensors with very small and custom shaped sensing elements. For example WO 2003/087801, describes biosensors and methods for producing biosensors for use in detecting and monitoring purines such as adenosine. These comprise a substrate comprising platinum or a platinum alloy; a first layer formed on the substrate comprising a sugar-derivative of a pyrrole and a second layer formed on the first layer comprising an amphiphilic pyrrole and one or more enzymes within the second layer. WO 2004/048603, describes methods of producing sol-gels on electrically conductive substrate to allow the entrapment of, for example, enzymes into a sol-gel matrix for the production of biosensors. Such micro-sensors are minimally invasive and have been used for detecting neurotransmitter release in the central nervous system. The microelectrodes are made from platinum or platinum alloy wire with all but about the final 2 mm of the Pt wire protected by a pulled glass capillary that is fused to the wire using heat. The exposed sensing tip suitably has a diameter from 25 to 100 um. These micro-sensors are fragile and the tips in particular require careful handling so as not to damage the sensing tip.
WO 2008/081193, incorporated herein, describes a biosensor comprising an electrically conductive substrate, with a first layer comprising Ruthenium Purple formed on the substrate, a second layer comprising polyaniline or a derivative thereof comprising one or more non-polar substituents formed on the first layer, and a third layer comprising one or more enzymes trapped within a matrix formed on the second layer. An advantage of using Ruthenium Purple, KFeRu(CN)6 or Fe4[Ru(CN)6]3, is that it allows small/micro biosensors to be formed, for example biosensors which are about 50 μm diameter and 0.1 to 2 mm in length. Preferred miniature biosensors are, for example less than 25 μm diameter and about 300 μm to about 2 mm long. Such biosensors are of particular use in the detection of analytes such as purines and derivatives thereof, particularly hypoxanthine. In operation, such biosensors are stable for several hours.